Microorganisms are widely used in the pharmaceutical industry to make products that are invaluable to human health such as drugs, their precursors, and biological agents such as vaccines, antibodies and hormones. Yeast is the organism of choice for these important applications due to the ease of its genetic manipulation, deep understanding of its basic biology, and the significant industrial advantages it has over other microorganisms. However, metabolic engineering efforts in yeast have focused mostly on the overexpression of enzymes in the cytoplasm, leaving the wealth of resources in its mitochondria essentially untapped. The goal of this project is to develop the tools, methods and strategies to engineer mitochondria in the yeast Saccharomysces cerevisiae, and gain access to the many benefits provided by this organelle for the production of new compounds important to the pharmaceutical and other industries. The first aim is to develop a new standardized vector series for protein expression in yeast (including targeted expression to mitochondria) to expedite and reduce the costs of cloning, assembly, troubleshooting and optimization of engineered pathways. This new vector series will help overcome many aspects of molecular biology that are often bottlenecks in any metabolic engineering project. The second aim is to demonstrate the benefits of mitochondrial engineering in the construction of heavy alcohol biosynthetic pathways, which stand to gain much by this new technology. This will involve the screening of pathway components, their assembly, and implementation using the new vector series described above. The third aim is to identify genes, mutations or conditions that affect mitochondrial physiology in favor of the engineered heavy alcohol biosynthetic pathways. This will entail the development of a new metabolic flux biosensor, which will allow the design of high throughput assays to screen thousands of strains rather than the few dozens that are feasible with current methods. The broad, long-term implication of this project is in laying the foundation for mitochondrial engineering, a new technology applicable to synthetic biology and metabolic engineering for countless benefits to health research and the pharmaceutical industry. PUBLIC HEALTH RELEVANCE: Baker's yeast, Saccharomyces cerevisiae, is a preferred microorganism for applications relevant to the pharmaceutical and other industries, including for the production of drugs and other biological agents such as vaccines. While the ease to carry out the genetic modifications required for these applications is one of the attributes that has made yeast such a useful organism, the wealth of resources provided by its mitochondria (the cell's powerhouse) remain essentially untapped for similar applications. This project focuses on the development of mitochondrial engineering to harness the unique advantages of this organelle, which will enable new yeast applications in synthetic biology and metabolic engineering to benefit health research and the pharmaceutical industry.